SIB2003 Molecular Biology Practicals
Manipulation and analysis of DNA using standard Molecular Biology Techniques.
During the course of the next three practical classes you shall be performing a number of techniques in order to isolate and manipulate DNA from bacteria. The practicals are spread over three sessions, the techniques that you will perform are indicated below:
Practical 1 Isolation of plasmid DNA from three cultures of E.coli using a method known as the alkaline lysis method.
Practical 2
(Part A) Digestion of the plasmid DNA that you have isolated, with restriction enzymes.
Practical 2
(Part B) Transformation of bacterial cells (E. coli) with:
a) No DNA
b) Plasmid DNA
c) digested DNA
Practical 3 Analysis of DNA from practicals 1 and 2 using the technique of agarose gel electrophoresis and analysis of transfomed E. coli from practical 2 (part B)
Whilst the practical classes deal with different techniques, it is important that you write it up as a single laboratory report. The write-up should contain a general introduction written as an overview. The methods and results should be written up as separate sections within the single write-up and the conclusion should be a general summing up exercise where you can discuss the relative success of each practical. Please remember that the write-up should be written up in the passive and in past tense. Further guidelines for the write-up are provided separately and uploaded on Unilearn.
Practical 1
DNA forensics is a division of forensic science that focuses on the use of genetic material in criminal investigation to answer questions pertaining to legal situations, including criminal and civil cases. Through DNA testing, law enforcement officers are able to identify human remains or the individual responsible for a crime. DNA testing is a highly advanced scientific process that involves replicating the human DNA sequence to create a genetic map of an individual. Because of its reliability, DNA testing has become a significant factor in criminal cases. However, it has also been identified as having the potential to violate privacy and constitutional rights. The DNA identification process consists of five stages. These five stages
Each human being has something called DNA. DNA is described as genetics and an extremely long macromolecule that is the main component of chromosomes and is the material that transfers genetic characteristics in all life forms. DNA constructs of two nucleotide strands coiled around each other in a ladder like arrangement with the sidepieces composed of alternating phosphate and deoxyribose units and the rungs composed of the purine and pyrimidine bases adenine, guanine, cytosine, and thymine. Each chromosome consist of one continuous thread-like molecule of DNA coiled tightly around proteins and contains a portion of the 6,400,000,000 basepairs that make up your DNA.
In this investigation pUC19 plasmids were used as the vector due to its small size of 2686bp, high uptake efficiency by the host and fast replication time. Important features of this plasmid include the origin of replication and multiple cloning sites (MCS). The origin of replication allows the plasmid to replicate inside the host bacterium. The MCS is located within the lacZ gene and contains unique sites for the Xbal & EcoRI restirction enzymes to cut and produce sticky ends for the CIH-1 gene to bind to. Furthermore, the pUC19 plasmid also contains an ampiccilin resistance gene so only transforemed E.coli are able to remain viable when spread on the agar plates that also has the addition of ampiccilin. The lacZ gene encodes the β-galactosidase enzyme which aids in indentifying the recombinant E.coli from the non recombinant cells (Coventry University 2016).
This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.
DNA. The isolated plasmids are ran on 40 ml 1% agarose gel at 110 volts with 1.2 μl 6X track
DNA is a term that has been used in science as well as in many parts of daily
The goal of this experiment was to investigate genetic transformation of E.coli through the reaction of organism to the vector pGLO plasmid. As mentioned, the pGLO plasmid contains genes coding for resistance to ampicillin (amp), and genes coding for production of the green fluorescent protein (GFP) which glows under UV light in the presence of arabinose (ara),which serves as a reporter gene. This green fluorescent
Next the tubes were placed in an ice bath, while obtaining a sterile loop to swipe a single colony of E.coli to put into the tube. After gently swiping a colony onto the loop, it was then spun in the +pGLO tube to get it to come off, returned to the ice bath. Next using a different sterile loop, it was swooped it in a container labeled pGLO plasmid DNA and again spun it ONLY into the tube with the solution labeled +pGLO to get it to come off. After about 10 minutes on ice the tubes were then placed into a 42ºC water bath for 50 seconds exactly, and immediately after placed them back into the ice bath. Finally, after 2 more minutes in the ice bath the tubes were separated into 4 containers. 250 ul of +pGLO solution was added to the containers containing +pGLO, LB broth, with ampicillin and +pGLO, LB broth, ampicillin, with arabinose. Also 250 ul of the –pGLO solution was added to the 2 containers containing LB broth, and ampicillin, with LB broth. Using a sterile loop for each plate the solutions were spread out gently and thoroughly on to the containers with agar. After the containers were incubated in 37ºC for at least 24 hours, the results were observed and disposed of (Weedman,
Transformation is the directed modification of a genome by the external application of DNA from a cell of different genotype (Griffiths and Miller).Bacterial transformation is the easiest type of transformation to create and study due to the single cellularity of bacteria and its
The results of our lab did not go as planned for the co-transformation. Specifically, the plates that used both plasmids were killed off by the anti-biotics, proving that both plasmids were not able to be transferred into the e. coli.
The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.
After the incubation, 1.5 mL of each of the three cultures were added to eppendorf tubes and centrifuged at 13,200 rpm for 1 minute. An alkaline lysis procedure like that of Birnboim and Doly was then performed to extract the plasmid DNA with 200 μl of alkaline SDS detergent solution (Birnboim & Doly, 1979). After
Let the test tube sit undisturbed for 2 - 5 minutes. You should begin to see air bubbles form at the boundary line between the ethanol and the filtered fruit solution. Bubbles will form near the top, and you will eventually see the DNA float to the top of the ethanol. Gently insert the stir stick into the test tube. Slowly raise and lower the tip several times to spool and collect the DNA. If there is an insufficient amount of DNA available, it may not float to the top of the solution in a form that can be easily spooled or removed from the tube. However, the DNA will still be visible as white/clear clusters by gently stirring the solution and pushing the clusters around the top. Post-Lab Questions What is the texture and consistency of the DNA DNA is viscous and greasy. Why did we use a salt in the extraction solution High salt makes DNA less soluble in water. In order to dissolve, the water needs to interact with the DNA. Since DNA is quite large, it needs to interact with lots of water for this purpose. When you add salt, the water preferentially interacts with the salt (its small, and can move around in solution easier than the DNA can). This makes it so there is less water available to interact with the DNA and it becomes less soluble. Is the DNA soluble in the aqueous solution or alcohol DNA is less soluble in an alcohol such as isopropanol than it is in water. This is because alcohols are non-polar, whereas water is polar. The polar
The chemical and reagents used for the extraction and quantitation of DNA were: Plant DNAzol (0.3ml/0.1g), 100% ethanol (100%: 0.225 ml/0.1 g, 75%: 0.3 ml/0.1 g), Chloroform (0.3 ml/0.1 g), Plant DNAzol-ethanol solution: Plant DNAzol, 100% ethanol (1:0.75 v/v), TE buffer (10 mM Tris, 1 mM EDTA pH 8.0), 1.2% agarose gel (Agarose, 1X TAE buffer), 6X loading buffer (glycerol, Tris/EDTA pH 8.0, ethidium bromide), .25X TAE buffer, Restriction enzymes and Restriction endonuclease buffers. All the chemicals used were quality grade. The restriction
Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double